Histamine-activated,non-selective cation currents and Ca2+ transients in endothelial cells from human umbilical vein

Permeation properties and modulation of an ionic current gated by histamine were measured in single endothelial cells from human umbilical cord veins by use of the patch-clamp technique in the ruptured-whole-cell mode or using perforated patches. We combined these current measurements with a microfluorimetric method to measure concomitantly free intracellular calcium concentration ([Ca²⁺]_i). Application of histamine induced an intracellular calcium transient and an ionic current that reversed near 0 mV. The amplitude of the current ranged from –0.2 to –2nA at –100mV. The tonic rise in [Ca²⁺]_i and the ionic current are partly due to Ca²⁺ influx. This Ca²⁺ entry pathway is also permeable for Ba²⁺ and Mn²⁺. The amplitude of the histamine-activated current was also closely correlated with the amplitude of the concomitant Ca²⁺ transient, suggesting that the latter is at least partially due to Ca²⁺ influx through histamine-activated channels. The reversal potential of the histamine-induced current was 7.6±4.1 mV (n=14) when the calcium concentration in the bath solution ([Ca²⁺]_o) was 1.5mmol/l. With 10 mmol/l [Ca²⁺]_o it was –13.7±4.7 mV and shifted to +13.0±1.5 mV in nominally Ca²⁺-free solution (n=3 cells). The amplitude of the current in Ca²⁺-free solution was enhanced compared to that in 10 mmol/l [Ca²⁺]_o. The shift of the reversal potential and the concomitant change of the current amplitude suggest that the channel is permeable for calcium but has a smaller permeability for calcium than for monovalent cations. The latency between the application of histamine and the appearance of the current was voltage dependent and was much smaller at more negative potentials. This effect is unlikely to be due to desensitization, but may suggest a voltage-dependent step in the signal transduction chain. Similar histamine-induced Ca²⁺ signals were observed if the currents were measured in patches perforated with nystatin. The onset of the agonist-activated current was, however, much more delayed and its amplitude significantly lower than in ruptured patches. The histamine-induced currents and intracellular Ca²⁺-transients were largely reduced after incubation of endothelial cells with the phorbol ester TPA. H7, a blocker of protein kinase C, induced membrane currents and Ca²⁺ signals in the absence of an agonist. It is concluded that the agonist-activated Ca²⁺-entry in endothelial cells occurs through non-selective cation channels which can be down-regulated by protein kinase C activation.     

Na/Ca exchangers in collecting cells of rat kidney

Single pieces of fura-2-loaded cortical collecting tubule (CCT) isolated either from normal or adrenalectomized (ADX) rats were superfused in vitro, and the cytosolic calcium concentration ([Ca²⁺]_i) was calculated from fluorescence recordings. The effects of altering the sodium gradient across cell membranes were investigated. Switching external sodium from 164 mM to 27 mM (low [Na⁺]_o) had little effect on [Ca²⁺]_i in normal tubules (106±9 versus 101±9 nM, n=15) whereas it resulted in a large peak of [Ca²⁺]_i in CCT from ADX-rats (270±32 versus 135±11 nM, n=21). Since CCT from ADX rats are known to have a reduced Na-pump activity, the effect of ouabain treatment on CCT from normal rats was also tested. When CCT from normal rats were exposed to 1 mM of ouabain in the presence of 164 mM of [Na⁺]_o, [Ca²⁺]_i increased only moderately (123±15 versus 111±11 nM, n=13); when the low [Na⁺]_o solution was applied to these ouabain-treated tubules, a large and transient increase in [Ca²⁺]_i was obtained (287±38 versus 123±15 nM, n=13). This response was absent with [Ca²⁺]_o=0. The data suggest the presence of 3 Na⁺/1 Ca²⁺ exchangers in cell membranes of rat CCT. The calcium flux equation derived by Läuger for the exchanger indicates a non-linear relationship between net calcium flux and driving force which could account for the difference observed here between the poor effect of applying either low [Na⁺]_o or ouabain alone and the large peak of [Ca²⁺]_i induced by combining these two conditions.     

Single channel Ca2+ currents inHelix pomatia neurons

Unitary Ca²⁺ currents of TEA injected Helix neurons were recorded in the Giga seal situation (6, 7) from microscopic membrane patches exposed to 50 mM [Ca²⁺]_o, O [Na⁺]_o, 20 mM [TEA⁺]_o and 2.5 M [TTX]_o. Constant field assumptions yield a channel permeability of 2.9±1.0×10^–14 cm³s^–1 corresponding to slope conductances of 5 to 15 pS between 0 and –30 mV. Frequency of occurrence of the units strongly increased with depolarization. Mean open time of the Ca²⁺ channels was about 3 ms without obvious dependence on voltage. A similar open time was seen with [Ba²⁺]_o, yielding about double the current strength when compared with [Ca²⁺]_o.     

Thapsigargin discharges intracellular calcium stores and induces transmembrane currents in human endothelial cells

We have measured the effects of thapsigargin, a specific inhibitor of endoplasmic Ca²⁺-adenosine 5-triphosphatase (Ca²⁺-ATPase), on membrane currents and on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in single endothelial cells from the human umbilical cord vein. Currents were recorded by means of the patchclamp technique in the whole-cell mode and [Ca²⁺]_i was measured using Fura II. Application of thapsigargin at concentrations between 0.2 and 2 mol/l induced a slow increase in [Ca²⁺]_i to a peak value of 400±110 nmol/l above a resting level of 120±35 nmol/l, and then slowly declined to a new steady-state level of 315±90 nmol/l (n=33). The thapsigargin-induced increase in [Ca²⁺]_i depended on the extracellular Ca²⁺ concentration ([Ca²⁺]_o: it declined after removal of extracellular Ca²⁺, but increased again when [Ca²⁺]_o was augmented, indicating that the response depends on a transmembrane influx of Ca²⁺ ions. The peak amplitude of the histamine-induced Ca²⁺ transient was reduced in the presence of thapsigargin. This reduction was more pronounced when histamine was applied at the peak of the increase in [Ca²⁺]_i induced by thapsigargin than during the rising phase of the changes in [Ca²⁺]_i. The decline of the Ca²⁺ transient induced by histamine after washing out the agonist was also affected by thapsigargin. Before application of thapsigargin, this decline could be described by a single exponential with a time constant equal to 24.5±5 s (n=7). In the presence of thapsigargin, the decline was much slower (n =8 cells), although in four cells a fraction of about 23% still exchanged with a similar fast value of 29.4±4 s. Thapsigargin also induced a slowly developing inward current in endothelial cells at a holding potential of –40 mV. Voltage ramps applied before and during the development of this current indicated that a non-selective cation channel with a reversal potential near 0 mV was activated. In contrast with the Ca²⁺ transients, these currents did not show a declining phase. These results indicate that inhibition of the endoplasmic Ca²⁺ pump in endothelial cells increases [Ca²⁺]_i. The tonic component of this increase might be partly due to opening of non-selective Ca²⁺-permeable cation channels activated by depletion of intracellular stores.     

Intracellular calcium in mammalian brain cells: fluorescence measurements with quin2

Summary Dispersed brain cells from 12–14 day old mouse embryos were loaded with the Ca²⁺-sensitive fluorescent probe, quin2 and shown to have a resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) of 158 nM (SE ± 5) in the presence of 1 mM [Ca²⁺]_o. When external [Ca²⁺] was raised from 0 to 1 mM there was an increase of [Ca²⁺]_i of 70 nM; with further additions of Ca to >10 mM [Ca²⁺]_o the level of [Ca²⁺]_i increased by <25 nM. Releasable intracellular Ca²⁺ stores, estimated from the increase in [Ca²⁺] produced by 4Br A23187 in the absence of extracellular Ca²⁺, were 24 fmol/10⁶ cells. A small increase in [Ca²⁺]_i could be produced by the mitochondrial inhibitor, carbonyl cyanide m-chlorophenylhydrazone (CCCP). When extracellular K⁺ was raised by 10–20 mM, intracellular Ca²⁺ levels increased from 152 (SE ± 7) to 204 nM (SE ± 10). These K⁺-induced increases in [Ca²⁺]_i were blocked by verapamil, did not occur in the absence of extracellular Ca²⁺, and presumably reflect the activation of voltage-dependent Ca²⁺ channels. N-methyl-D-aspartic acid (NMDA) evoked an increase in [Ca²⁺]_i, while the kainate-like lathyrus sativus neurotoxin, L-3-oxalyl-amino-2aminopropionic acid (L-3,2-OAP) did not; this is consistent with previous observations of different and respectively Ca²⁺-dependent and -independent mechanisms of action of these excitatory amino acids.     

Electrophysiological properties of membrane currents in single myometrial cells isolated from pregnant rats

The whole-cell voltage-clamp method was applied to single smooth muscle cells prepared from the longitudinal layer of the pregnant rat myometrium (17–20 days of gestation). It was found that the transient inward current mainly consists of Ca²⁺ current, because the removal of Ca²⁺ ions from the external medium and 10 M nifedipine eliminated this inward current. Its steady-state inactivation curve was obtained by the standard method, in which the membrane potential of half inactivation and the slope factor were estimated to be –58.0±4.9 mV (n=11) and 8.9±1.4 mV (n=11), respectively. In a small number of preparations (in 2 out of 30 preparations), there remained a very fast inward current in Ca²⁺-free medium containing Mg²⁺. Tetrodotoxin (TTX, 10 M) can abolish this current, suggesting that the channel for this current is equivalent to the Na⁺ channel in nerve cells. Two major phases of outward currents were identified by voltage jumps from negative holding levels to more positive levels. The first phase was a fast transient outward current. This current remained intact after external tetraethylammonium (TEA, 20 mM) was added. Following the transient current, a large delayed rectified outward current reached its peak over a period of 50 ms and then decayed. The reversal potential for this outward current was determined by observing the change of polarity of the tail currents with the change in extracellular K⁺ concentration ([K⁺]₀). The slope for the change of reversal potential per ten-fold change in [K⁺]₀ is 57.7 mV at more than 23.2 mM [K⁺]_o, indicating that this current is mostly carried by K⁺ ions. Voltage-dependent inactivation of the delayed rectified outward current was determined by the standard method. The membrane potential for half inactivation and the slope factor were estimated to be –42.8±3.9 mV (n=3) and 10.1±1.5 mV (n=3), respectively. External TEA (20 mM) effectively eliminated the delayed rectified outward currents. Nifedipine (10 M) suppressed not only Ca²⁺ current but also outward K⁺ currents.     

Intracellular Na+ modulates large conductance Ca2+-activated K+ currents in human umbilical vein endothelial cells

We studied the effects of Na⁺ influx on large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in cultured human umbilical vein endothelial cells (HUVECs) by means of patch clamp and SBFI microfluorescence measurements. In current-clamped HUVECs, extracellular Na⁺ replacement by NMDG⁺ or mannitol hyperpolarized cells. In voltage-clamped HUVECs, changing membrane potential from 0 mV to negative potentials increased intracellular Na⁺ concentration ([Na⁺]_i) and vice versa. In addition, extracellular Na⁺ depletion decreased [Na⁺]_i. In voltage-clamped cells, BK_Ca currents were markedly increased by extracellular Na⁺ depletion. In inside-out patches, increasing [Na⁺]_i from 0 to 20 or 40 mM reduced single channel conductance but not open probability (NPo) of BK_Ca channels and decreasing intracellular K⁺ concentration ([K⁺]_i) gradually from 140 to 70 mM reduced both single channel conductance and NPo. Furthermore, increasing [Na⁺]_i gradually from 0 to 70 mM, by replacing K⁺, markedly reduced single channel conductance and NPo. The Na⁺–Ca²⁺ exchange blocker Ni²⁺ or KB-R7943 decreased [Na⁺]_i and increased BK_Ca currents simultaneously, and the Na⁺ ionophore monensin completely inhibited BK_Ca currents. BK_Ca currents were significantly augmented by increasing extracellular K⁺ concentration ([K⁺]_o) from 6 to 12 mM and significantly reduced by decreasing [K⁺]_o from 12 or 6 to 0 mM or applying the Na⁺–K⁺ pump inhibitor ouabain. These results suggest that intracellular Na⁺ inhibit single channel conductance of BK_Ca channels and that intracellular K⁺ increases single channel conductance and NPo. GH Liang and MY Kim contributed equally to this publication and therefore share the first authorship.     

Actions of pinacidil on membrane currents in canine ventricular myocytes and their modulation by intracellular ATP and cAMP

We studied the effects of pinacidil (3–50 M) on the membrane currents of canine ventricular myocytes, using the whole-cell variant of the patch-clamp technique, and the modulation of these effects by intracellular environment, using the pipette perfusion technique. The following observations were obtained: (1) pinacidil induced a dosedependent outward shift in current at voltages positive to ±70 mV; (2) the pinacidil-induced current was largely timeindependent at voltages positive to ±50 mV and displayed an increase in current fluctuations at more positive voltages, resembling the kinetic properties of current through the ATP-regulated K⁺ channels; (3) elevating the extracellular potassium concentration ([K⁺]_o) caused a positive shift in the voltage where the pinacidil-induced current crossed the voltage axis and increased the slope conductance of this current; (4) the pinacidil-induced current was reduced by Ba²⁺ (0.5–1.5 mM) and abolished by intracellular Cs⁺ (125 mM); (5) glibenclamide reversibly reduced or abolished the pinacidil-induced current; (6) the action of pinacidil was decreased by elevating [ATP] in the pipette solution (from 1 to 10 mM); (7) the action of pinacidil was augmented by adding isoproterenol (1 M) to the superfusate or adding cAMP (0.1 mM) to the pipette solution; (8) elevating temperature augmented, and accelerated the onset of, pinacidil's action; (9) pinacidil reversibly decreased the Ca²⁺ -independent transient outward current (I_to1) but augmented the Ca²⁺ -dependent transient outward current (I_to2). Based on these observations, we reached the following conclusions: (1) the main effect of pinacidil is to increase an outward current through the ATP-regulated K⁺ channels; (2) pinacidil's action is modulated by an enzymatic reaction.     

Potassium and calcium currents and action potentials in mouse Balb/c 3T3 fibroblasts

The electrical properties of Balb/c 3T3 mouse fibroblasts were studied with the whole-cell patch clamp technique. In current clamp mode a resting potential of —75.5±2.1 mV was recorded. In voltage clamp mode an inward current was also observed at potentials negative toV _m. This current crossed the 0-current axis at a voltage nearV _m, and rectified at more positive potentials; the degree of rectification was dependent on [K⁺]_o. At potentials positive to –30 mV a transient inward current was observed, showing a peak amplitude of –193±36 pA at+10 mV; the current amplitude was dependent on voltage and [Ca²⁺]_o, it was strongly increased by 20 mM BaCl₂ and abolished by 2 M verapamil and 1 M nifedipine. These cells, in response to depolarizing stimuli, develop slow action potentials, probably supported by the Ca²⁺ current.     

Phasic changes in intracellular pH during action potentials of sheep Purkinje fibres

Regulation of intracellular pH (pH_i) and the relationship between H⁺ and Ca²⁺ may vary during activity. Ion-selective microelectrodes were used to record pH_i during action potentials of sheep Purkinje fibres prolonged by low temperature (21°C) and elevated CO₂ content. Intracellular pH also was measured during changes in extracellular calcium concentration, [Ca²⁺]_o. Cytosolic alkalinization (peak pH_i change, 0.03–0.05) was observed during the long action-potential plateau and transient acidification (0.01–0.02 units) upon repolarization. Potassium-induced depolarization to plateau potentials (i.e. to –15±2 mV) simulated the peak magnitude of the alkalinization. However, compensation for the alkalinization occurred at a faster rate during the action potential (8.9±4.3 nM/min) than during K⁺ depolarization (1.2±0.5 nM/min). In comparison, the cytoplasm acidified in resting fibres (0.06–0.07 log units) during changes of [Ca²⁺]_o thought to increase intracellular calcium concentration. Alterations of pH_i were translated into changes of proton concentration ([H⁺]_i). Ten-to twenty-fold elevation of [Ca²⁺]_o evoked a comparable change in [H⁺]_i (mean increase, 5.7 nM) but oppositely directed from that during the plateau (mean decrease, 8.8 nM). The findings in resting fibres seem consistent with displacement of bound protons by Ca²⁺. In contrast, the initial change in pH_i during the plateau is proposed to be consequent to Ca²⁺-release from sarcoplasmic reticulum and/or phosphocreatine hydrolysis coupled to ATP regeneration.     

Electrogenic Na+/K+-transport in human endothelial cells

Na⁺/K⁺ pump currents were measured in endothelial cells from human umbilical cord vein using the whole-cell or nystatin-perforated-patch-clamp technique combined with intracellular calcium concentration ([Ca²⁺]_i) measurements with Fura-2/AM. Loading endothelial cells through the patch pipette with 40 mmol/l [Na⁺] did not induce significant changes of [Ca²⁺]_i. Superfusing the cells with K⁺-free solutions also did not significantly affect [Ca²⁺]_i. Reapplication of K⁺ after superfusion of the cells with K⁺-free solution induced an outward current at a holding potential of 0 mV. This current was nearly completely blocked by 100 mol/l dihydroouabain (DHO) and was therefore identified as a Na⁺/K⁺ pump current. During block and reactivation of the Na⁺/K⁺ pump no changes in [Ca²⁺]_i could be observed. Pump currents were blocked concentration dependently by DHO. The concentration for half-maximal inhibition was 21 mol/l. This value is larger than that reported for other tissues and the block was practically irreversible. Insulin (10–1000 U/l) did not affect the pump currents. An increase of the intracellular Na⁺ concentration ([Na⁺]_i) enhanced the amplitude of the pump current. Half-maximal activation of the pump current by [Na⁺]_i occurred at about 60 mmol/l. The concentration for half-maximal activation by extracellular K⁺ was 2.4±1.2 mmol/l, and 0.4±0.1 and 8.7±0.7 mmol/l for Tl⁺ and NH₄ ⁺ respectively. The voltage dependence of the DHO-sensitive current was obtained by applying linear voltage ramps. Its reversal potential was more negative than –150 mV. Pump currents measured with the conventional whole-cell technique were about four times smaller than pump currents recorded with the nystatin-perforated-patch method. If however 100 mol/l guanosine 5-O-(3-thiotriphosphate) (GTPS) were added to the pipette solution, the currents measured in the ruptured-whole-cell-mode were not significantly different from the currents measured with the perforated-patch technique. We suppose that the use of the perforated-patch technique prevents wash out of a guanine nucleotide-binding protein (G-protein)-connected intracellular regulator that is necessary for pump activation.     

Calcium receptor message, expression and function decrease in differentiating keratinocytes

Calcium-sensing receptor (CaSR) expression and function were studied in proliferating and differentiating cultured human gingival keratinocytes (HGKs). CaSR mRNA and protein were present in proliferating HGKs cultured in 0.03 mM [Ca²⁺] and decreased in cells induced to differentiate by culturing in 1.2 mM [Ca²⁺] for 2 days. CaSR protein was also detected in gingival tissue. Exposure to 10 mM extracellular [Ca²⁺] activated two sequential whole-cell currents. The first was a small, transient calcium release activated calcium current I_CRAC-like current with an inwardly rectifying I-V curve. The second current was larger with a linear I-V curve. Both currents were significantly decreased in differentiating cells. Neither neomycin nor gadolinium induced changes in whole cell currents nor in intracellular [Ca²⁺], but neomycin inhibited the late large current. Extracellular Ca²⁺ increased intracellular [Ca²⁺] of proliferating HGKs in a dose-dependent fashion. Comparison of the time-courses of the whole-cell currents and the intracellular [Ca²⁺] responses indicated both induced currents supported a Ca²⁺ influx. Extracellular [Mg²⁺] changes did not affect intracellular [Ca²⁺]. La³⁺ and 2-APB inhibited the whole cell current and intracellular [Ca²⁺] changes. The results indicate that the CaSR signaling response likely plays a major role in initiating Ca²⁺ induced differentiation responses in HGKs.     

[Ca2+]i-dependent membrane currents in guinea-pig ventricular cells in the absence of Na/Ca exchange

Transient inward currents (I _ti) during oscillations of intracellular [Ca²⁺] ([Ca²⁺]_i) in ventricular myocytes have been ascribed to Na/Ca exchange. We have investigated whether other Ca²⁺-dependent membrane currents contribute to I _ti in single guinea-pig ventricular myocytes, by examining membrane currents during [Ca²⁺]_i oscillations and during caffeine-induced Ca²⁺ release from the sarcoplasmic reticulum in the absence of Na⁺. Membrane currents were recorded during whole-cell voltage clamp and [Ca²⁺]_i measured simultaneously with fura-2. In the absence of Na/Ca exchange, i.e., with Li⁺, Cs⁺ or N-methyl-D-glucamine (NMDG⁺) substituted for Na⁺, the cell could be loaded with Ca²⁺ by repetitive depolarizations to +10 mV, resulting in spontaneous [Ca²⁺]_i oscillations. During these oscillations, no inward currents were seen, but instead spontaneous Ca²⁺ release was accompanied by a shift of the membrane current in the outward direction at potentials between –40 mV and +60 mV. This [Ca²⁺]_i-dependent outward current shift was not abolished when NMDG⁺ was substituted for internal monovalent cations, nor was it sensitive to substitution of external Cl^–. It was however, sensitive to the blockade of I_Ca by verapamil. These results suggest that the transient outward current shift observed during spontaneous Ca²⁺ release represents [Ca²⁺]_idependent transient inhibition of I _Ca. Similarly, during the [Ca²⁺]_i transients induced by brief caffeine (10 mM) applications, we could not detect membrane currents attributable to a Ca²⁺-activated nonselective cation channel, or to a Ca²⁺-activated Cl^– channel; however, transient Ca²⁺-dependent inhibition of I _Ca was again observed. We conclude that neither the Ca²⁺-activated nonselective cation channel nor the Ca²⁺-activated Cl^– channel contribute significantly to the membrane currents during spontaneous [Ca²⁺]_i oscillations in guineapig ventricular myocytes. However, in the voltage range between –40 mV and +60 mV Ca²⁺-dependent transient inhibition of I _Ca will contribute to the oscillations of the membrane current.     

Voltage-dependent Ca2+ influx in the epithelial cell line HT29: simultaneous use of intracellular Ca2+ measurements and nystatin perforated patch-clamp technique

Indirect evidence has accumulated indicating a voltage dependence of the agonist-stimulated Ca²⁺ influx into epithelial cells. Manoeuvres expected to depolarise the membrane voltage during agonist stimulation resulted in: (1) a decrease of the sustained phase of the adenosine triphosphate (ATP, 10^–5 mol/l)-induced intracellular Ca²⁺ transient, (2) a reduced fura-2 Mn²⁺-quenching rate, and (3) prevention of the refilling of the agonist-sensitive store. To quantify the change in intracellular Ca²⁺ as a function of membrane voltage, we measured simultaneously the intracellular Ca²⁺ activity ([Ca²⁺]_i) with fura-2 and the electrical properties using the nystatin perforated patch-clamp technique in single HT₂₉ cells. Ca²⁺ influx was either stimulated by ATP (10^–5 mol/l) or thapsigargin (TG, 10^–8 mol/l). After [Ca²⁺]_i reached the sustained plateau phase we clamped the membrane voltage in steps of 10 mV in either direction. A stepwise depolarisation resulted in a stepwise reduction of [Ca²⁺]_i. Similarly a stepwise hyperpolarisation resulted in a stepwise increase of [Ca²⁺]_i (ATP: 27.5±10 nmol/l per 10 mV, n=6; TG: 19 ±7.9 nmol/l per 10 mV, n=12). The summarised data show a linear relationship between the fluorescence ratio 340/380 nm change and the applied holding voltage. In unstimulated cells the same voltage-clamp protocol did not change [Ca²⁺]_i (n=9). Under extracellular Ca²⁺-free conditions [Ca²⁺]_i remained unaltered when changing the membrane voltage. These data provide direct evidence that the Ca²⁺ influx in epithelial cells is membrane voltage dependent. Our data indicate that small changes in membrane voltage lead to substantial changes in [Ca²⁺]_i. This may be due either to a change of driving force for Ca²⁺ into the cell, or may reflect voltage-dependent regulation of the respective Ca²⁺ entry mechanism.     

Actions of growth-hormone-releasing hormone on rat pituitary cells: intracellular calcium and ionic currents

Actions of growth-hormone-releasing hormone (GHRH) on single rat anterior pituitary cells were studied using indo-1 fluorescence to monitor changes in intracellular calcium, [Ca²⁺]_i, and perforated-patch recording to measure changes in membrane potential and ionic currents. GHRH elevated [Ca²⁺]_i in non-voltage-clamped cells by a mechanism that was dependent upon extracellular Na⁺ and Ca²⁺ and was blocked by the dihydropyridine Ca²⁺-channel blocker, nitrendipine. Resting cells had a fluctuating membrane potential whose a mean value depolarized by 9 mV in response to GHRH. The membrane-permeant cAMP analogue, 8-(4-chlorophenylthio)cAMP, mimicked the action of GHRH on membrane potential. Under voltage clamping, GHRH activated a small inward current (1–5 pA). Two types of response could be distinguished. The type I response had an inward current that was largest at more negative potentials (–90 mV), and the type II response had inward current that was larger at more positive potentials (–40 to –70 mV). Both types of response were reversible and blocked by removal of extracellular Na⁺. These results suggest that the rise in [Ca²⁺]_i produced by GHRH in non-voltage-clamped cells results from the activation via cAMP of a Na⁺-dependent conductance, which depolarizes the cell and increases the Ca²⁺ influx through voltage-gated Ca²⁺ channels.Dedicated in memory of the late Alexander P. Naumov.     

Free cytosolic calcium during spontaneous contractions in smooth muscle of the guinea-pig mesotubarium

The free intracellular calcium ion concentration ([Ca²⁺]_i) was measured simultaneously with isometric force in strips of guinea-pig mesotubarium using the Fura-2 technique. During the relaxed period (5–15 min) between spontaneous contractions [Ca²⁺]_i continues to decrease after full mechanical relaxation to reach a minimal level of 86±8 nM (n=9) just before the start of the next contraction. During the spontaneous contractions (5–15 min) [Ca²⁺]_i reached a maximum of 211±19 nM and then oscillated between 155±16 nM and 194±9 nM. Increased extracellular Ca²⁺ concentration to 10 mM from the standard concentration of 1.5 mM caused a decreased frequency of spontaneous contractions and an increase in [Ca²⁺]_i both in the relaxed and contracted states. In 10 mM extracellular Ca²⁺, addition of AlF₄ ^–, as 1 mM NaF + 10 M AlCl₃, caused a sustained increase in [Ca²⁺]_i and maintained force. Addition of verapamil (10 M) in this situation decreased [Ca²⁺]_i to the resting level. The results suggest that the cyclic appearance of trains of action potentials is related to variation in [Ca²⁺]_i, possibly via inactivation of Ca²⁺-dependent K⁺ channels.     

Extracellular magnesium regulates intracellular free Mg2+ in vascular smooth muscle cells

Summary Regulatory effects of extracellular magnesium ions ([Mg²⁺]_o) on intracellular free ionized magnesium ([Mg²⁺]_i) were exmained in cultured vascular smooth muscle cells (VSMCs) fromrat aorta by digital imaging microscopy using the Mg²⁺ fluorescent probe, Mag-fura-2. With normal Mg²⁺(1.2 mM)-containing incubation media, [Mg²⁺]_i in VSMCs was 0.63±0.09 mM. The ratio of [Mg²⁺]_i/[Mg²⁺]_o was 0.52±0.07. Elevation of [Mg²⁺]o up to 4.8 mM induced consistent increments in [Mg²⁺]_i (to a mean values of 1.63±0.08 mM) in 5 min and lowered the ratio of [Mg²⁺]_i/[Mg²⁺]_o to 0.34±0.02. Our data suggest that [Mg²⁺]_o can regulate [Mg²⁺]_i, which may be related to its effects on intracellular Ca²⁺ ([Ca²⁺]_i) and tone of VSMCs.     

The dihydropyridine niguldipine inhibits T-type Ca2+ currents in atrial myocytes

The whole-cell tight seal recording technique was used to investigate the effects of niguldipine, a novel dihydropyridine, on Ca²⁺ currents in guinea pig atrial cells. Ca²⁺ currents were separated into T-type and L-type components by an appropriate voltage protocol. Extracellular application of 1 M (±)-niguldipine (NIG) resulted in a pronounced blockade of both T-type (to 20±10 % of control, n=5) and L-type Ca²⁺ currents (to 28±12 % of control, n=5). Current to voltage relationships clearly showed that both Ca²⁺ currents were blocked over the whole voltage range examined (-60 to +40 mV). The inhibitory effect of niguldipine on T-type Ca²⁺ currents was found to be voltage-dependent, i. e. prolonged hyperpolarization to -90 mV led to a partial and transient removal of NIG block. The IC₅₀ for T-type Ca²⁺ current inhibition by (±)-NIG was determined as 0.18 M. NIG action is stereospecific. (+)-niguldipine was found to be more potent than (-)-niguldipine in blocking both Ca²⁺ currents. This study demonstrates the Ca²⁺ antagonistic action of the dihydropyridine NIG, which may not discriminate between T- and L-type Ca²⁺ channels.     

Cytosolic free calcium in single microdissected rat cortical collecting tubules

Cytosolic free Ca²⁺ ([Ca²⁺]_i) was measured in single fragments of rat cortical collecting tubule (CCT) by using fura-2 and a tubule superfusion device. Under basal conditions, i.e. with 1 mM of external Ca²⁺ ([Ca²⁺]_o), the average steady state [Ca²⁺]_i was 179±16 nM (n=44 tubules). Random alterations of [Ca²⁺]_o between 0 mM and 4 mM led to corresponding variations in steady state [Ca²⁺]_i levels, which were linearly correlated with [Ca²⁺]_o (average slope 93±34 nM [Ca²⁺]_i per 1 mM [Ca²⁺]_o for six tubules). In contrast, [Ca²⁺]_i was little affected by decreasing external Na⁺ concentration. Cell membrane depolarization with 100 mM of external K⁺ induced a sustained drop in [Ca²⁺]_i (21% as an average). The data suggest that steady state [Ca²⁺]_i in CCT cells resulted from a non-saturable passive entry of calcium ions across cell membranes balanced with an active extrusion by calcium ATPase (pump and leak mechanism). The passive component cannot be accounted for either by Na⁺/Ca²⁺ exchangers nor by voltage-dependent calcium channels; it is best explained by the presence of voltage-independent calcium channels in cell membranes.     

Swelling of rat mesangial cells induces a Ca2+-dependent Cl− conductance

Membrane voltage (V _m) and ion currents of rat mesangial cells in primary culture were measured with the patch-clamp technique in the fast whole-cell configuration.V _m was –44 ± 1 mV (_n = 138). A reduction of the osmolality from 290 to 190 mosmol/kg depolarizedV _m from –44 ± 1 to –29 ± 1 mV (_n = 118) and increased the inward and outward conductances (Gm) from 14±2 to 39 ± 4 nS and 13±2 to 37 ± 4 nS (_n = 84), respectively. During the hypotonicity-induced depolarization the cell capacitance increased significantly from 33 ± 3 to 42 ± 4 pF (_n = 40). The effect of hypotonic cell swelling onV _m was increased in a bath with a reduced extracellular Cl^– of 32 mmol/l (by 71 ± 4%,_n = 23), indicating that a Cl^– conductance was activated. The permselectivity of this conductance was I^– Br^– > Cl^–. TheV _m response was not affected in the presence of a reduced extracellular Na⁺ of 5 mmol/l (n = 13) and was inhibited in a solution with reduced extracellular Ca²⁺ concentration (by 63 ± 9%,n = 14). In microfluorescence measurements with the Ca²⁺-sensitive dye fura-2 hypotonic cell swelling induced a sustained increase of the intracellular Ca²⁺ activity, [Ca²⁺]_i (_n = 19). The increase of [Ca²⁺]_i was completely inhibited when the extracellular solution was free of Ca²⁺. TheV _m response to hypotonic cell swelling was not attenuated in the presence of the L-type Ca²⁺ channel blockers nicardipine (n = 5), nifedipine (n = 5) and verapamil (n = 5) (all at 1 mol/l). The data indicate that in rat mesangial cells, osmotic swelling induces a Ca²⁺ influx from extracellular space. This Ca²⁺ influx activates a Cl^– conductance resulting in a depolarization ofV _m. The enhanced Cl^– conductance may lead to KCl extrusion and hence regulatory volume decrease.